User Manual - PCR Array 2.0 , Inc.
Contents
1. 28 15 Data analysis Using ACt Method c ccccccccssssssssseeececessesseceeeesseeseaeeeeeeecesscusnseaeeeesesseesenaaas 32 16 2 FAQs and TroublesHOOtin gs a a A a aE e Sivvek sls AAEE R CSAR 35 User s Manual qMethyl DNA Methylation Detection qPCR System 3 General Information qMethy products are covered by a limited warranty A copy of the warranty is included with this manual Appendix to be added The operator may be required to store received product under specified conditions as described herein to keep the warranty in effect Information in this document is subject to change without notice PCRarray 2 0 Inc assumes no responsibility for any errors that may appear in this document This document is believed to be complete and accurate at the time of publication In no event shall PCRarray 2 0 Inc be liable for incidental special multiple or consequential damage in connection with or arising from the use of this product qMethyl product is designed for research use only and is not intended for use in diagnostic procedures Use of the qMethy products conveys no patent rights expressly or by implication under any patent or patent application owned or licensed by PCRarray 2 0 Inc that covers qMethyl products Specifically but without limitation no right immunity authorization or license is granted expressly or by implication for the usage of qMethyl technology Limitation of Liabilit2. Additional Materials amp Equipment Required A DNA Isolation Kit See our Recommendations in the Protocol Section qMethyl Restriction Enzyme Kit EM 03 MANDATORY for a Complete DNA digestion qlight SYBR Green qPCR Master Mixes MANDATORY for a Complete and Successful Experiment Note Be sure to pick the correct one for the instrumentation in your laboratory Master mix Type Cat No Size Real Time PCR cyclers 2X 96 well plates ee 200 rxn 2X1 3mL rxn 3m P All ABI amp Stratagene qLight ROX SYBR 12X 96 well plates Instrumentation Eppendorf green Master Mix MR1 12 MR1 200 rxn 12X1 3mL Mastercycler ep realplex Instruments with ROX filter set 24X 96 well plates MR1 24 200 rxn 24X1 3mL l 2X 96 well plates l MEHE 200 rxn 2X1 3mL gLight rxn 3m Fluorescein SYBR Meo 12X 96 well plates BioRad iCylcer MyiQ and iQ5 Green Master Mix 200 rxn 12X1 3mL Instrumentation MF2 24X 96 well plates MF2 24 200 rxn 24X1 3mL MM3 02 AE E BioRad Opticon Opticon 2 amp qLight SYBR Cae aoe Chromo 4 Roche LightCycler Green Master Mix Ringe 12X 96 well plates 480 System Eppendorf MM3 200 rxn 12X1 3mL Mastercycler ep realplex No reference dye Instruments without ROX filter 24X 96 well plates MM3 24 set 200 rxn 24X1 3mL User s Manual qMethyl DNA Methylation Detection qPCR System 1 2 3 4 5 1 2 3 4 5 44 Gene 96 Well PCR Arrays F
3. Setup and run the PCR reactions for the restriction digests 1 Prepare PCR reaction cocktails In two reservoirs prepare the PCR cocktails with the two DNA digests for each sample as below Components MoEmix MsE mix 1 ddH 0 40W 450 2 PCR Master mix 550 ul 550 ul 3 MoE digest 100 ul 4 MsE digest 100 ul Total 1100W 11004 2 Mix well by shaking gently 3 Use 12 channel pipette to add 10 ul of MoE cocktail to each well of rows from A to H then add 10 ul of MsE cocktail similarly to each wells of rows from to P O 5 Ny Sample 1 Sample 2 4 Seal the plate wells spin down briefly 2000 rpm for 1 minute 23 User s Manual qMethyl DNA Methylation Detection qPCR System 5 To run the PCR reactions use the two step cycling program shown bellow for all of PCR cyclers Cycles Temperature Duration 1 95 C 10 minutes 98 C 15 seconds 40 75 C 1 minute Add melting curve segment Optional Important note The 10 minute step at 95 C is required to activate the HotStart DNA polymerase Detect and record SYBR Green fluorescence from every well during the annealing step of each cycle Step 3 Data analysis Refer to section D for Data analysis 24 User s Manual qMethyl DNA Methylation Detection qPCR System 13 Protocol 5 384 well plate PCR array X1 for 1 sample 188 gene assay jm Step 1 Setup restriction digestion using qMethy
4. all the left DNA in the two digests not affected by the complex configuration of the methylation patterns A locus consisting of 24 CpG sites contains 16 777 216 2 possible different methylation configurations Making primer and probe sets capable of monitoring all of them which may be also biological or pathological relevant would seem to be impossible Thus for MSP based PCR methods the primers pick up only small fraction of converted templates whose methylation configurations are complementary to designed primers so the sensitivity is greatly reduced if the CpG site methylation of target DNA sequences is not 100 Guaranteed high specificity and amplification efficiency for GC rich sequences We have successfully achieved this with our validated primer design criteria and computer algorithm the great master mix and the universal PCR reaction parameters Primers for unconverted DNA are much easier to be designed than those for MSP using our unique primer design software for the high GC rich sequences Usually MSP primers with high amplification efficiency and specificity can not be obtained before trial and error optimization of PCR reactions 4 4 Why hypermethylated DNA copies can be reliably detected quantitatively with this technology The hypermethylated DNA copies are determined using DNA digested with methylation sensitive restriction enzyme The enzyme s recognition sequence contains a CpG dinucleotide When it is get
5. 2000 rpm for 1 minute 4 To run the PCR reactions use the two step cycling program shown bellow for all of PCR cyclers 5 Cycles Temperature Duration 1 95 C 10 minutes 98 C 15 seconds 40 75 C 1 minute Add melting curve segment Optional Important note The 10 minute step at 95 C is required to activate the HotStart DNA polymerase Detect and record SYBR Green fluorescence from every well during the annealing step of each cycle 29 User s Manual qMethyl DNA Methylation Detection qPCR System If you assay a gene on multiple samples set up the PCR reaction as below a For a 96 well block prepare the PCR cocktail without DNA samples first follow the table below sampleX2 sampleX4 sampleX8 sample 12 1 dd0 16nl 32W 64u pl 2 PCR Master mix 40 ul 80 ul 160 ul 240 ul 3 PCR primer mix 4ul 8 ul 16 ul 24 ul Total 60 160 240 360 For a 384 well plate prepare the PCR cocktail without DNA samples first follow the table below sampleX2 sampleX4 sampleX8 sample 12 1 dd0 8p l6 32W 48u 2 PCR Master mix 20 ul 40 ul 80 ul 120 ul 3 PCR primer mix 2 ul 4ul 8 ul 12 ul Total 30 ul 80 ul 120 ul 180 ul b For 96 well block add 15 of the cocktails to each PCR tube or well then add 5 ul of the MoE digests and MsE digests to each tube for a sample accordingly For 384 well plate add 7 5 ul of the cocktails to each well of the plate then add 2 5 ul the MoE digest and MsE digest for each samp
6. Contents oresar heh te ated ea aaa aa e RE 3 3 General INFORMATION of eaa sete i aa ee decade E Ea a a Eaa d EA RAE AA ARES 4 4 Warnings and Safety Precautions cccccccccccsssssssscecececeesessaeeeeecesseseaeseceeeceseeeseasaeeeesessenseaaees 5 Soo MIMBO CUCU OM aina aa aea a du ceed aa a a a ea E L a 6 62 Materials Provided csinos an i a a Eat 8 7 Additional materials amp equipment required ssssssssseessssssesesessssssrreersssssesereessessssssrrernessssene 9 8 Consideration before Starting the Experiment cccccsssscceceesssssaececeeecessesnsaeeceeeeseeseeseaaeas 11 9 Protocol 1 96 well plate PCR array X1 for 1 sample 44 gene assay ccecsesssseeeeeceseeeseees 13 10 Protocol 2 384 well plate PCR array X1 for 4 samples 44 gene ASSAY cscsssseceeeeeseeseraees 16 11 Protocol 3 96 well plate PCR array X 2 for 1 sample 88 gene aSSAY ccceesssseceeeceeenesnaees 19 12 Protocol 4 384 well plate PCR array X1 for 2 samples 92 gene ASSAY cscssssececeeeseesenaees 22 11 Protocol 3 96 well plate PCR array X 2 for 1 sample 88 gene aSSAY ccseesssseceeeeesenseraees 19 12 Protocol 4 384 well plate PCR array X1 for 2 samples 92 gene ASSAY cecssssececeeesessenaees 22 13 Protocol 5 384 well plate PCR array X1 for 1 sample 188 gene assay ccccscccceceeessentees 25 14 Protocol 6 Multiple sample assay of individual genes with a 96 well or 384 well plate
7. 2 0 or the Corbett Research Rotorgene Calibrated Multi Channel Pipettor RNase DNase free pipette tips and tubes RNase DNase free 100 ul regular PCR reaction tubes 8 or 12 tube strings Molecular biology grade ddH O RNase and DNase free 10 User s Manual qMethyl DNA Methylation Detection qPCR System 8 Consideration before Starting the Experiment IS important Please read through the entire procedure before proceeding to your experiment A Genomic DNA Preparation and Quality Control High quality DNA is ESSENTIAL for obtaining accurate results The most important prerequisite for the DNA methylation detection assay is consistent high quality genomic DNA from every experimental sample It is very helpful to follow the below recommendations 1 Genomic DNA preparation methods In general popular commercially available DNA purification products are good enough for sample DNA isolation Here we recommend the followings Cultured Cells Use 5 Primer s PerfectPure DNA purification kit Cat 2302000 Web www 5Prime com Tissue Samples Use Qiagen s QlAamp DNA Mini Kit manual or EZ1 DNA Tissue Kit automatic Cat 51304 amp 953034 Web www1 qiagen com Whole blood bone marrow samples Qiagen s QlAamp DNA Blood Mini Kit Manual or EZ1 DNA Blood 200 ul Kit automatic Cat 51104 51204 Web www1 qiagen com For Other Biological Samples Search literatures to find protocols to pre
8. The second Generation of PCR Array i Z PCRarray 2 0 lt 46400 Benedict Drive Ste 105 Sterling VA 20164 P 1 866 489 5499 F 1 703 637 9863 support pcrarray2 com www pcrarray2 com qMethyl DNA Methylation Detection qPCR System Simple Fast Reliable No Bisulfite Conversion Version 1 2 For Research Use Only Not For Use in Diagnostics User s Manual qMethyl DNA Methylation Detection qPCR System 1 Receiving Unpacking and Storing the Products We recommend that you inspect the package of the products immediately after opening the shipping container and make sure that there are no visible damages to the product Please contact PCRarray 2 0 Inc for assistance if there are observable irregularities Store the products according to the label on the package Typically we recommend that you store qMethyl product at 20 C for storage up to a 12 month period Do not open the product container prior use Water moisture may come in contact with the product and cause adverse effects on the performance SP vote We recommend that you immediately inspect the packages for possible visible damages due to shipment and store the packages under conditions according to the product label User s Manual qMethyl DNA Methylation Detection qPCR System 2 Table of Contents 1 Receiving Unpacking and Storing the Products cccccccccccssssssecececsesssesseaeceeeeeseseseseaeseeeeseesees 2 2 Table of
9. able for quantitative detection of heterogeneously methylated DNA populations therefore it is suitable for analysis of tissue sample consisting of mixed cell types This is attributed to 1 the high specificity sensitivity amp amplification efficiency of PCR reaction guaranteed by the specifically primer design criteria and reaction parameters 2 four selective digestion of DNA samples that include unique DNA methylation dependent restriction enzyme to eliminate background 3 comparable PCR results from the selective digests 4 specific and complete restriction digestion To get the details go through the answers to questions 2 5 How can it be guaranteed that the restriction digestion of the sample DNA be complete This is achieved by over digestion of DNA samples using longer time and 20 fold higher amount of the DNA methylation sensitive enzyme A unspecific digestion is negligible if there is any Ten cycle differences between MoE and MsE digest Ct values for 100 unmethylated DNA target it means 0 01 DNA are not digested or only 1 5 copies of DNA molecules remain undigested in the target DNA region when 5 ng is used A copy of monoploidy human genomic DNA is about 3 3333 pg How reliable are the results from the qMethyl qPCR assay The reliability of the technology is guaranteed by the following design principles 1 The guaranteed complete restriction digestion This is achieved by over digestion of DNA samples using long
10. c DNA of high quality from tissues of single cells types or from your sample DNA if 60 of the cells are target cells As in the cases of any bisulfite conversion based PCR methods the result variability will decrease significantly with increased amounts of input DNA So use larger amounts of input DNA as long as possible when clinical heterogeneous tissue samples are used For example when assay are performed to profile gene DNA methylation in cancer cell DNA amounts should be increased when heavy non tumor cell contaminations are expected such as stroma cells inflammation cells and blood cells etc In general we recommend the initial input DNA amount per target assay is 10 ng to 30 ng for 96 well blocks or 2 5 ng to 7 ng PCR reaction for 384 well block For an array of 44 gene assays we recommend use 0 6 ug DNA sample and for 188 genes assays with 384 well block use 1 5 ug DNA C Preparing a clean workspace free of DNA contamination For reliable results it is very important to avoid any contamination of the reactions with products of previous PCR experiments Please follow the recommendations below 1 Physically separate workspaces for PCR setup from that for post PCR performance Before experiment setup decontaminate PCR workspace working in a PCR operation hood is highly recommended 2 Wear gloves throughout the procedure Use only fresh PCR grade reagents and lab ware 3 Prepare dilutions Do not peel the protective fil
11. cycle Step 3 Data analysis Refer to section D for Data analysis 21 User s Manual qMethyl DNA Methylation Detection qPCR System 12 Protocol 4 384 well plate PCR array X1 for 2 samples 92 gene assay Step 1 Setup restriction digestion using qMethyl Restriction Enzyme Kit EM 03 1 Thaw the 5 X digestion buffer and dissolve the cotton like precipitates in by vortex briefly Then prepare cocktail without the enzymes in a sterilized 1 5 ml microtube for each sample following the table below We recommend using 1 5 2 5 ug DNA for each sample 2 Component Volume a ddo Xp 2 5XRM Buffer 42 ul 3 1 5 3 0 ug Sample DNA X ul Toa oo owa X Y 148 ul 3 Vortex at high speed briefly and spin down 4 Then set up the MoE and MsE digestion reactions in two 200 ul reaction tubes PCR for each sample following the table below Note This formula is to ensure that the two reactions contain equal amount of DNA Component MoE digestion1 MoE digestion 2 1 Digestion cocktail 93 ul 93 ul 2 Enzyme MoE 10 ul 10 ul 3 Enzyme MsE Total 103 ul 103 ul 5 Mix well by inverting the tubes up and down several times 6 Incubate at 37 C for 4 hours or overnight in any PCR thermal cycler 7 Inactivate the enzymes at 65 C for 20 minutes following the digestion Then store the DNA digests at 20 C for two years or 80 C indefinitely 22 User s Manual qMethyl DNA Methylation Detection qPCR System Step 2
12. each well from rows A to D of the 96 well plate 1 gene 1 to gene 44 and plate 2 gene 45 to gene 88 Aliquot 20 ul of MsE mix to each well from rows E to G of the above two 96 well plates Plate 1 Gene well locations mone ee ose le les eels A Gor coz cos coa os cos co7 cos cos c10 a11 G12 moet B 613 G14 Gis c16 G17 c18 G19 a20 Gas G22 623 G24 Reaction c G25 aze a27 c28 629 G30 a31 a32 a33 a34 35 c36 gt c37 cas c39 cao Gar Gaz cas caa oci ocz ocs pcre 20 User s Manual qMethyl DNA Methylation Detection qPCR System Plate 2 Gene well locations Reaction G69 670 a71 G72 a73 G7 a75 676 677 G78 679 cso cst Gs2 Gas cea cas ase c87 Gas pc ocz ocs Pcro A B Cc D 3 Seal or cap the wells Spin down the plates briefly 2000 rpm for 1 minute 4 To run one plate first with the two step program shown bellow for all cyclers Keep the remained one in 4 C and run it immediately after the first one is finished if you have only one machine Cycles Temperature Duration 1 95 C 10 minutes 98 C 15 seconds 40 75 C 1 minute Add melting curve segment Optional Important note 1 The 10 minute step at 95 C is required to activate the HotStart DNA polymerase Detect and record SYBR Green fluorescence from every well during the annealing step of each
13. eactions for the two restriction digests 1 With the two DNA digests prepare individual PCR cocktails in two 1 5 ml tubes or reservoirs of suitable size following the table below Component MoE mix MsE mix 1 ddH 0 490 ul 490 ul 2 PCR master mix 550 ul 550 ul 3 MoE digest 62 ul 4 MsE digest 62 ul Total 1102 ul 1102 pl 2 Mix well then load the mixes to a signature array plate you have ordered Aliquot 20 ul of MoE mix to each well from row A to D of the 96 well plate Aliquot 20 ul of MsE mix to each well from row E to G of the 96 well plate i Gene well locations Digest A cor co2 cos coa cos G06 Gor Gos Goo io cat 612 mot B 613 cm cas c16 617 c1s G19 620 621 622 G23 624 Reaction c a25 626 27 628 c29 630 G31 a32 c33 csa G35 c36 gt G37 c38 c39 cao cas Gaz Gas caa oca ocz ocs Pcre 3 Seal or cap the wells spin down the plates briefly 2000 rpm for 1 minute 4 Torun the reactions use the two step program shown bellow for all cyclers Cycles Temperature Duration 1 95 C 10 minutes 98 C 15 seconds 40 75 C 1 minute Add melting curve segment Optional 14 User s Manual qMethyl DNA Methylation Detection qPCR System Important note The 10 minute step at 95 C is required to activate the HotStart DNA polymerase Detect and record SYBR Green fluorescence from every well duri
14. ep 1 DNA Digestion 1 2 3 Mix DNA with the digestion buffer Divide the mix equally into two tubes Enzyme O Enzyme S 37 C 4 hours overnight Add the enzymes accordingly 4 Incubate at 37 C for 6 hours overnight 1 2 3 4 5 6 7 8 9 10 11 12 A 200000909 Step 2 Setup and run the PCR reaction ak pt pd gd gee det ged 1 Mix the two digests with PCR master mix Reaction E 80969890898 see A 200000000000 individually and load to the PCR plate 3999000099090009 mE OOOO0000080 Reaction c OOOOSOSSO08 2 Run the Real time PCR reactions 0000000000080 Step 3 Data analysis Pan Pane 1 Collect raw Ct data Geinciniele Deincishin Tiii Viie 2 Use analysis template to obtain Data analysis methylation data i 1 i Figure The simple three step assay Step 1 Mock DNA digestion wit Enzyme O and PCR MoE In the digestions enzyme blend that do not digest targeted genomic regions is added The PCR reactions measure the initial input DNA of the target regions Step 2 Methylation sensitive enzyme digestion of DNA with Enzyme S and PCR MsE In the digestion methylation sensitive restriction enzyme S is added so the unmethylated DNA will be cleaved while the methylated DNA remains intact The PCR reactions that follow measure the methylated DNA Step 3 Data analysis The hypermethylated and unmethylated DNA fractions in a sample are easily obtained simply by cutting and pasting the raw Ct
15. er time and 20 fold higher amount of the DNA methylation sensitive enzyme A unspecific digestion is negligible if there is any Also refer to the answer to question 2 Comparable results from the two digests For any given target sequences same primer pairs are used to amplify the two differential digests i e the PCR results are generated from same target DNA sequences and same primers the only difference is the intact DNA copy number left in the two digests 2 This is a great advantage over bisulfite modification based PCR methods For a given target with bisulfite methods two different primer pairs are used to amplify methylated and unmethylated templates from bisulfite conversion This will cause biased results due to different amplification efficiency of the two primer pairs In vitro methylated reference DNA and normalization reaction such as the one used in MethyLight real time PCR has to be used to correct this problem but it still requires that all the primer pairs show similar amplification efficiency This is a painful challenge that is achieved only after trial and error optimizations Further more inclusion of 34 User s Manual qMethyl DNA Methylation Detection qPCR System reference DNA also introduces artifacts caused by in consistent incomplete in vitro methylation of the DNA in addition to extra cost 3 Better sensitivity The better sensitivity comes from the facts that the same pair primer amplify
16. he results The plots of the results will appear in the Data Chart sheet two plots will appear 1 Hypermethylated genes 2 Unmethylated genes for each sample Homozygous deletions When the Ct values from all the four digests for a gene are equal to or larger than 32 genomic homozygous deletion is highly suggested at the locus e g in cancer cell genomic DNA To validate such results checking the PCR products is recommended by gel electrophoresis no single dominant bands between 75 bp and 125 bp should be observed The significance of the results Cutoff value setting The cutoff for the number of methylated DNA copies is set up by users to indicate if the sample is positive for the gene methylation The same principles used in bisulfite based real time PCR methods apply here too You may set your cutoff based on the following principles In most cases only hypermethylated promoters cause transcription repression of cognate genes so the cutoff is usually set at 5 15 hypermethylated DNA i e at or above that level it is consider that methylation is positive for the genes The stringency is dependent on the extent of non target cell contamination This is similar to using bisulfite based PCR methods experimental design 33 User s Manual qMethyl DNA Methylation Detection qPCR System 16 FAQs and Troubleshooting 1 Is the technology reliable for heterogenous tissue samples This technology is highly desir
17. l Restriction Enzyme Kit EM 03 1 Thaw the 5 X digestion buffer and dissolve the cotton like precipitates in by vortex briefly Then prepare cocktail without the enzymes in a sterilized 1 5 ml microtube following the table bellow We recommend using 4 0 ug DNA Component Volume 1 ddH20 X ul 2 5X RM Buffer 84 ul 3 3 0 6 0 ug Sample DNA Y ul Total 380 ul X Y 93 5 ul 2 Vortex at high speed briefly and spin down 3 Then set up the MoE and MsE digestion reactions each in two 200 ul PCR reaction tubes following the table bellow Note This formula is to ensure that the two reactions contain equal amount of DNA Component MoE digestion1 MoE digestion2 MsE digestion 1 MsE digestion 2 1 Digestion cocktail 90nl 90W W w t 2 Enzyme MoE 10 ul 10 ul 3 Enzyme MsE 10 ul 10 ul Total 100 ul 100 ul 100 ul 100 ul 4 Mix well by inverting the tubes up and down several times 5 Incubate at 37 C for 4 hours or overnight in any PCR thermal cycler 25 User s Manual qMethyl DNA Methylation Detection qPCR System 6 Inactivate the enzymes at 65 C for 20 minutes following the digestion Then store the DNA digests at 20 C for two years or 80 C indefinitely Step 2 Setup PCR reactions for the restriction digests 1 Prepare PCR reaction cocktails In two reservoirs prepare the PCR cocktails with the two DNA digests as below Components MoEmix MsE mix 1 ddH 0 90W 9004 2 PCR Maste
18. le the wells c Seal the plate wells or cap the tubes spin down briefly 2000 rpm for 1 minute d To run the PCR reactions use the two step cycling program shown bellow for all of PCR cycler Cycles Temperature Duration 1 95 C 10 minutes 98 C 15 seconds 40 75 C 1 minute Add melting curve segment Optional 30 User s Manual qMethyl DNA Methylation Detection qPCR System Step 3 Data analysis Refer to section D for Data analysis 31 User s Manual qMethyl DNA Methylation Detection qPCR System 15 Data analysis using ACt Methods Note 4 The PCR array analysis template in Excel format can be downloaded from the link at http www PCRarray2 com qMethylPCRArray Plate php Click the PCR Array Data Analysis link found in the lower Product Support section of the gray right hand sidebar It will make the analysis very easy Obtaining threshold cycle values After the PCR reaction obtain threshold cycle C values following instructions of the used instruments We recommend manually setting the Baseline and Threshold Value 1 1 To define the Baseline use the Linear View of the amplification plots and set the instrument to use the readings from cycle number 3 through the cycle which is before the earliest visible amplification This is often cycle 15 1 2 To define the Threshold Value use the Log View of the amplification plots and place it above the background signal but within the lo
19. low We recommend using 1 5 2 5 ug DNA for each sample Component Volume 1 ddH20 X ul 2 5X ResBuffer 13 ul 3 0 1 0 2 ug sample DNA Yul Total 60 ul X Y 47 ul 2 Vortex at high speed briefly and spin down 3 Then set up the MoE and MsE digestion reactions in two 200 ul reaction tubes PCR for each sample following the table below Note This formula is to ensure that the two reactions contain equal amount of DNA Component MoE digestion MsE digestion 1 Digestion cocktail 299 29 l 2 Enzyme MoE 2 5 ul O ul 3 Enzyme MsE O ul 2 5 ul Total 3145W 8S 4 Mix well by inverting the tubes up and down several times 5 Incubate at 37 C for 4 hours or overnight in a PCR thermal cycler at volume 30 ul 28 User s Manual qMethyl DNA Methylation Detection qPCR System 6 Inactivate the enzymes at 65 C for 20 minutes following the digestion Then store the DNA digests at 20 C for two years or 80 C indefinitely Step 2 Setup and run the PCR reactions for the restriction digests 1 Prepare PCR reaction cocktails In two real time PCR reaction tubes a 96 well PCR plate ora 384 well plate prepare the PCR cocktails with the two DNA digests for each sample as below Components MoE mix MsE mix 1 ddH 0 Sul Sul 2 PCR Master mix 10 ul 10 ul 3 Primer mix 10 uM 1 ul 1 ul 4 MoE digest 4ul 5 MsE digest 4 ul Total 20 ul 20 ul 2 Mix well by shaking gently 3 Seal the plate wells spin down briefly
20. lve the cotton like precipitates in by vortex briefly Then prepare cocktail without the enzymes following the table bellow We recommend using 4 ug DNA Component Volume 1 ddH20 X pl 2 5XRM Buffer 42 ul 3 1 5 3 0 ug Sample DNA Y ul Total 190 ul X Y 148 ul 2 Mix well by vortex and spin down briefly 3 Then set up the MoE and MsE digestion reactions in two 200 ul PCR reaction tubesfollowing the table bellow Note This formula is to ensure that the two reactions contain equal amount of DNA Component MoE digestion MsE digestion _ 1 Digestion cocktail 90 ul 90 ul 2 Enzyme MoE 10 ul 3 Enzyme MsE 10 ul Total 100W 100 ul 4 Mix thoroughly by inverting the tubes up and down several times 5 Incubate at 37 C for 4 hours or overnight in a thermal cycler 6 Inactivate the enzymes at 65 C for 20 minutes following the digestion Store DNA digests at 20 C till use Spin down before use 19 User s Manual qMethyl DNA Methylation Detection qPCR System Step 2 Setup PCR reactions for the four restriction digests 1 Prepare PCR cocktails prepare the PCR cocktails with the two DNA digests in two reservoirs as below Component 1 ddH 0 2 PCR master mix 3 MoE digest 4 MsE digest Total 2 Mix well by vortex and spin down briefly Then l MoE mix 1000 ul 1100 ul 100 ul 2200 ul MsE Mix 1000 ul 1100 ul 100 ul 2200 ul Aliquot 20 ul of MoE mix to
21. m from the PCR array plate until immediate use 4 Close all tubes containing PCR products once you finish solution transfer 12 User s Manual qMethyl DNA Methylation Detection qPCR System 9 Protocol 1 96 well plate PCR array X1 for 1 sample 44 gene assay Step 1 Setup restriction digestion using qMethyl Restriction Enzyme Kit EM 03 1 Thaw the 5 X digestion buffer and dissolve the cotton like precipitates in by vortex briefly Then prepare cocktail without the enzymes following the table bellow we recommend using 1 ug DNA Component Volume 1 ddH 0 X pl 2 5X ResBuffer 26 5 ul 3 0 6 1 2 ug sample DNA Y ul Total 120 ul X Y 93 5 ul 2 Mix well by vortex and spin down briefly 3 Then set up the MoE amp MsE digestion reactions in two 200 ul PCR reaction tubes following the table below Note This formula is to ensure that the two reactions contain equal amount of DNA Component MoE digestion MsE digestion 1 Digestion cocktail 58 ul 58 ul 2 Enzyme MoE Sul O ul 3 Enzyme MsE O ul Sul Total 63 ul 63 ul 4 Mix thoroughly by inverting the tubes up and down several times 5 Incubate at 37 C for 4 hours or overnight in a thermal cycler 6 Inactivate the enzymes at 65 C for 20 minutes following the digestion Store DNA digests at 20 C till use Spin down before use 13 User s Manual qMethyl DNA Methylation Detection qPCR System Step 2 Setup PCR r
22. methylated the cut will be blocked The recognition sequence occurs in over 98 CpG islands located in gene promoters In the amplicon design as many as possible such sites are included and only a small fraction of the amplicons contain one such restriction site Only when the amplicon sequences are densely methylated can all these sites also get methylated and block cutting the amplicons Thus after Enzyme A digestion only densely methylated amplicon sequences are left intact and detected Such design is representational based on the fact that in most cases only hypermethylated promoters are relevant to silencing gene transcription In the design only one or a few CpG sites within a DNA methylation target sequence are analyzed and their methylation status are used as reporter signal if target regions are densely methylated or not it is widely used such as in restriction digestion based methods including Southern blot MSRE and restriction landmark genomic screening RLGS as well as bisulfite conversion based methods such as methylation specific PCR MSP TaqMan based MethyLight MS SNuPE GoldenGate DNA Methylation BeadArray Infinium DNA Methylation BeadChip and COBRA etc 5 What cause incomplete restriction enzyme digestion Incomplete digestion will affect the sensitivity significantly Complete digestion of DNA sample are guaranteed with EpiQ Restriction Digestion Kit and the protocol refer to the answer to question 2 The most comm
23. ng the annealing extension step of each cycle Step 3 Data analysis with raw Ct values see below Section D 15 User s Manual qMethyl DNA Methylation Detection qPCR System 10 Protocol 2 384 well plate PCR array X1 for 4 samples 44 gene assay Step 1 Setup restriction digestion using qMethyl Restriction Enzyme Kit EM 03 1 Thaw the 5 X digestion buffer and dissolve the cotton like precipitates by vortex briefly Then prepare cocktail without the enzymes following the table bellow We recommend using 0 5 ug DNA For each sample Component Volume 1 ddH O0 X ul 2 5X ResBuffer 26 5 ul 3 0 3 0 8 ug sample DNA Y pl Total 120 ul X Y 93 5 ul 2 Mix well by vortex and spin down briefly 3 Set up four digestion reactions following the table bellow Note This formula is to ensure that the two reactions contain equal amount of DNA Component MoE digestion MsE digestion 1 Digestion cocktail 58 pl 58 ul 2 Enzyme MoE 5 ul Oul 3 Enzyme MsE Oul Sul Total 634 63 ul 4 Mix thoroughly by inverting the tubes up and down several times 5 Incubate at 37 C for 4 hours or overnight in a thermal cycler 16 User s Manual qMethyl DNA Methylation Detection qPCR System 6 Inactivate the enzymes at 65 C for 20 minutes following the digestion Store DNA digests at 20 C till use Spin down before use Step 2 Setup PCR reactions for the two restriction digestion products 1 In
24. on reasons are DNA samples of poor quality Sometime poor DNA quality is encountered when prepared from primary tissues and sometime it is difficult to get DNA samples of 35 User s Manual qMethyl DNA Methylation Detection qPCR System high quality from some organ tissues such as adult lungs from heavy smoking individuals Several common reasons are 1 Low enzyme activity The restriction enzymes used are expired much lower amount of the enzymes is used McrBC will be expired after 6 months while Hhal will expire after two years Before use always check the label of the enzyme microtubes for the expiration date DNA samples have heavy RNA contamination This will inhibit DNA digestion with restriction enzymes and will also cause significant inaccuracy in DNA quantification Be sure to include any RNase treatment steps in recommended DNA isolation procedures See Page B 3 Too much DNA is used due to inaccurate quantification or miscalculation Use right methods and instruments to quantify DNA samples Impure DNA when DNAs are prepared from organ tissues that are difficult for DNA isolation protein and or polysaccharide contamination may occur and will inhibit restriction enzyme activity significantly Use recommended DNA isolation kits refer to Section IV Part A for genomic DNA preparation 5 Incorrect incubation refer to Section IV Part B 6 Organic reagents such as chloroform phenol isopropanol are used in pre
25. ormat A B C D E Use the pack sizes of master mix described in the above table 88 Gene 96 Well PCR Arrays Format A B C D E Each a set of 2 array packs requires two 2 of the correct master mix for your instrument 1 X MR 02 MF 02 MM 02 44 Gene 384 Well PCR Arrays Format F G Each 384 well PCR Array 4 pack Formats F G requires four 4 of the correct master mix for your instrument 2 X MR 02 MF 02 MM 02 92 gene 384 well PCR arrays Format F G The two 2 twelve 12 and twenty four 24 pack sizes Formats F amp G require a quantity of two 2 of the correct master mixes for your instrument of the size for the corresponding 96 well PCR Array packs 2 X MR 02 MF 02 MM 02 or 2 X MR 12 MF 12 MM 12 or 2 X MR 24 MF 24 MM 24 188 Gene 384 Well PCR Arrays Format F G The two 2 twelve 12 and twenty four 24 pack sizes Formats F amp G require a quantity of two 2 of the correct master mixes for your instrument of the size for the corresponding 96 well PCR Array packs 2 X MR 02 MF 02 MM 02 or 2 X MR 12 MF 12 MM 12 or 2 X MR 24 MF 24 MM 24 Additional Equipment amp Reagents For recommendations on specific real time instrumentation thermal cyclers with fluorescent detection see the list of master mixes and plate formats above NOTE The PCR Arrays can only be used in 96 well and 384 well real time PCR instruments PCR Arrays can not be used in the Cepheid SmartCycler the Roche LightCycler
26. paration of DNA and the reagents are not completely removed etc Whenever possible avoid using these enzyme activity inhibiting organic solvents for DNA isolation 2 4 6 High Ct values from mock treated undigested DNA products 1 The initial denaturation step 95 C for 10 min is not used to activate the Taq polymorphism 2 The amounts of DNA used are too low 1 miscalculation 2 incorrect quantification of sample DNA 3 heavy RNA contamination Check your calculations and use correct methods and instruments to isolate and quantify DNA sample DNA is degraded DNA samples are contaminated by microbes due to improper storage of your DNA samples such as at 4 C Always store your DNA samples at 20 C more than 2 years and at 80 C indefinitely 4 Improper storage of primer preloaded qPCR array plates at high temperature too long 5 Loss of activity of the real time PCR Master Mix due to improper storage possibly left at high temperature for too long 3 7 Will pipetting error affect the PCR Array results The passive reference dyes in the PCR master mixes such as ROX and Fluorescein and other technologies such as for Roch Lightcycler 480 are used by the real time PCR systems to normalize variation from well to well These normalization design combined with our specific digestion and PCR reaction setup protocol design the performance system can tolerate minor volume variations caused b
27. pare DNA of high quality from special biological samples or contact a Technical Support representative For best results all DNA samples should be suspended in DNase free water or alternatively in DNase free 10 mM Tris buffer pH 8 0 2 Measure DNA concentration and purity by UV spectrophotometer Prepare dilutions and measure absorbance in DNase free 10 mM Tris pH 8 0 buffer The spectral properties of nucleic acids are highly dependent on pH Concentration by A260 should be greater than 4 yg ml total DNA A260 A280 ratio should be greater than 1 8 A260 A230 ratio should be greater than 1 7 11 User s Manual qMethyl DNA Methylation Detection qPCR System 3 Avoid contamination of DNA samples Whatever DNA isolation methods or commercial kits you may use DO NOT forget to add RNase to remove RNA Otherwise heavy RNA contamination of DNA samples will cause inaccurate DNA quantification and affect DNA digestion efficiency 4 DNA quality control Assay For best results from the PCR array all DNA samples should also demonstrate consistent high quality Use our DNA sample quality control kit Cat to assay your DNA sample quality The assay kit provides control DNA samples and primers for you to test the quality of your DNA samples in parallel For detailed information go to our website B Sample DNA amount considerations for an assay For any single gene assay the method can yield reliable data with 4 ng genomi
28. r mix 1100 ul 1100 ul 3 MoE digest 200 ul 4 MsE digest 200 ul Total 2200W 2200 l 2 Mix well by shaking gently 3 Use 12 channel pipette to add 10 ul of MoE cocktail to each well of rows from A to H then add 10 ul of MsE cocktail similarly to each wells of rows from to P G y G a pa m 2 B ERE BEREaESSEESEE 26 User s Manual qMethyl DNA Methylation Detection qPCR System 4 Seal the plate wells spin down briefly 2000 rpm for 1 minute 5 To run the PCR reactions use the two step cycling program shown bellow for all of PCR cyclers Cycles Temperature Duration 1 95 C 10 minutes 98 C 15 seconds 40 75 C 1 minute Add melting curve segment Optional Important note The 10 minute step at 95 C is required to activate the HotStart DNA polymerase Detect and record SYBR Green fluorescence from every well during the annealing step of each cycle Step 3 Data analysis Refer to section D for Data analysis 27 User s Manual qMethyl DNA Methylation Detection qPCR System 14 Protocol 6 Multiple sample assay of individual genes with a 96 well plate or 384 well plate J Step 1 Setup restriction digestion using qMethyl Restriction Enzyme Kit EM 03 1 Thaw the 5 X digestion buffer and dissolve the cotton like precipitates in by vortex briefly Then prepare cocktail without the enzymes in a sterilized 1 5 ml microtube for each sample following the table be
29. ted region reactivates the transcription It is a fast growing research field in molecular biology particularly in cancer epigenetic research field Our qMethyl gene methylation system is a SYBR green real time PCR based detection system after complete digestion of sample DNA with optimized methylation sensitive restriction enzyme buffer system The whole procedure is very simple fast and reliable with much less genomic DNA compared to all bisulfite conversion based methods A The Benefits 1 Simple amp fast In two hours with just 0 5 to 1 ug genomic DNA for one sample up to 44 target genes can be detected in one run with 96 well PCR plate or 188 genes with 384 well PCR plate Reliable assay design All the assays are specifically designed based on established methylated genomic region for known genes Complete digestion guaranteed complete digestion of sample DNA with the optimized buffer enzyme system Robust quantitative PCR performance The PCR primers and cycling parameters are specifically designed for very high GC rich content all the PCR reactions are validated High sensitivity 20 methylated DNA copies can be reliably detected in 5ng genomic DNA in one reaction Free easy to use online Data analysis software within one minute all genes in one run can be analyzed with the exported raw Ct values User s Manual qMethyl DNA Methylation Detection qPCR System B The Outline of Simple Three step Assay St
30. two 1 5 ml tubes prepare PCR cocktails with the two DNA digests from each sample following the table below Components MoE Mix MsE Mix 1 ddH0 198m 198 yl 2 PCR Master mix 260 ul 260 ul 3 MoE digest 62 ul 4 MsE digest 62 ul Total 520W 520 ul 2 Mix well by vortex and spin down briefly 3 Add 10 ul of the PCR mixes for each sample to the proper wells in the 384 well plate following the guidance shown in the figure below 6 8 10 12 14 16 18 20 22 13 17 21 23 24 Sample 1 Sample 2 Sample 3 4 Seal the wells and spin down briefly Sample 4 17 User s Manual qMethyl DNA Methylation Detection qPCR System 5 To run the PCR reactions use the two step cycling program shown below for all of PCR cyclers Cycles Temperature Duration 1 95 C 10 minutes 98 C l 15 seconds 40 75 C 1 minute Add melting curve segment Optional Important note The 10 minute step at 95 C is required to activate the HotStart DNA polymerase Detect and record SYBR Green fluorescence from every well during the annealing step of each cycle Step 3 Data analysis Refer to section D for Data analysis 18 User s Manual qMethyl DNA Methylation Detection qPCR System 11 Protocol 3 96 well plate PCR array X 2 for 1 sample 88 gene assay Step 1 Set up Restriction Digestion using qMethyl Restriction Enzyme Kit EM 03 1 Thaw the 5 X digestion buffer and disso
31. values into the proper cells of the FREE qMethyl DNA Methylation Data Analysis Excel Template See the instructions in the excel for detail It is developed based on ACt method with MoE reaction Ct values as initial DNA input signals and those from MsE reaction as hypermethylated signal User s Manual qMethyl DNA Methylation Detection qPCR System 6 Materials Provided qMethyl array plates are provided based on the following format Format Real Time Instruments Plate All ABI standard blocks 7000 7300 7500 7700 7900 A Bio Rad iCycler MyiQ 96 well Bio Rad MJ Research Chromo 4 StepOnePlus ABI 7500 and 7900 FAST 96 well blocks 96 well Stratagene Mx3005p Mx3000p 96 well Bio Rad MJ Research D Opticon and Opticon 2 96 well Stratagene Mx4000 E Roche LightCycler 480 96 well block 96 well F ABI 7900 384 well block 384 well G Roche LightCycler 480 384 well block 384 well NOTE The format of a PCR array is indicated by the last digit of the catalog number Be sure that you have the correct PCR array format for your instrument before starting the experiment For example If Cat No HMETO10 12A is ordered twelve qMethyl plates of format A will be shipped Each PCR array shipment includes the arrays and either twelve 12 optical thin wall 8 cap strips Formats A amp D or one 1 optical adhesive film Formats B C E F amp G per array User s Manual qMethyl DNA Methylation Detection qPCR System
32. wer third of the linear phase of the amplification plot Export the Ct values Export the Ct values to a blank Excel spreadsheet following the instruction for your instruments Perform data analysis with the Ct values and data analysis templates for your assay from our website following the instructions in Instruction worksheet shown in the data analysis templates Data quality analysis 3 1 MoE Ct values The MoE Ct values for all genes should be within the range of 23 to 29 if 5 10 ng DNA are used The amplification efficiencies for all primer pairs are larger than 85 3 2 MSE The Ct values in MsE will be same as those of MoE s or higher than those of MoE s depending on the methylated DNA copy numner in the samples 3 3 Enzyme digestion efficiency estimation In QC Data report worksheet of data analysis template excel workbook check the DC control for digestion completeness The ACt value between average Ct values of the 3 DC controls and those of the MoE values should be larger than 5 Result analysis 4 1 Relative copy numbers of unmethylated hypermethylated DNA For each assay columns DM amp UM in the Results worksheet show the relative copy numbers of 32 User s Manual qMethyl DNA Methylation Detection qPCR System 4 2 4 3 4 4 hypermethylated methylated unmethylated DNA species accordingly For detailed information on the calculation of these values refer to appendixes The plots of t
33. y Except to the extent prohibited by local law in no event will PCRarray 2 0 Inc be liable for the direct indirect or other damages arising out of the use inability to use or the results of using qMethy products The use of these products is entirely at your own risk User s Manual qMethyl DNA Methylation Detection qPCR System 4 Warnings and Safety Precautions The following precautions should be followed to minimize the possibility of personal injury and or damage to property while using qMethy I products All users should observe good laboratory practices GLP Examples of GLP include a b c d Wearing of lab safety equipment including gloves eye protection and lab coat when preparing samples Do not pipette by mouth do not eat or drink in areas where specimens are being handled Clean any spill immediately Disposing of all specimens controls and materials used in testing as though they contain infectious agents User s Manual qMethyl DNA Methylation Detection qPCR System 5 Introduction DNA methylation is the enzymatic addition of a methyl group to cytosines in CG dinucleotide DNA sequences often at CpG islands CGI s without changing primary DNA sequences Such modifications at gene regulatory regions in particular promoters correlate well with the transcriptional state of the cognate gene hypermethylation represses transcription while demethylation of previous methyla
34. y pipetting errors Even a 20 pipetting error causes only 0 05 cycles of Ct change 7 How can I prevent the evaporation of reaction volume from the wells Be sure to carefully and completely seal the PCR Array with the optical thin wall 8 cap strips or the optical adhesive film before placing it into your thermal cycler 36 User s Manual qMethyl DNA Methylation Detection qPCR System If you have additional questions please check our website www PCRarray2 com for a more complete listing of Frequently Asked Questions FAQs or call our Technical Support Representatives at 1 888 XXX XXXX or XXX XXXX 37
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